PROJECT SUMMARY Kidney stone disease is highly prevalent, increasingly common, and associated with considerable morbidity. However, no new treatments to prevent kidney stones have been introduced in the last 30 years. Understanding how dysbiosis of the gut microbiome contributes to nephrolithiasis could lead to novel new treatments for kidney stone prevention. However, most prior studies of the gut microbiome in this population focused on Oxalobacter formigenes without considering the role of the entire gut microbiome in the gut-kidney axis, which is the complex interplay between the intestinal and urinary tracts in human health and disease. A critical barrier to developing new treatments for stone prevention is a lack of understanding of how perturbations of the gut microbiome and downstream changes in metabolites in the intestinal and urinary tracts contribute to kidney stone disease. In this proposal, we build on our recent discoveries of the role of diet, antibiotics, the gut microbiome, and the metabolome in kidney stone disease. We leverage an interdisciplinary team that is uniquely poised to define the human gut-kidney axis in kidney stone disease by combining expertise in using nutritional profiling, mediation analyses of high-dimensional microbiome and metabolomic data, and large data analytics. The proposed research tests the central hypothesis that diet and antibiotics contribute to nephrolithiasis by perturbing the gut- kidney axis through alterations of the gut microbiome. In doing so, the proposed studies will identify metabolic pathways in the gut-kidney axis that could be targets for novel therapeutics to prevent kidney stones. In Aim 1, we will identify perturbations of the microbiome and metabolome in kidney stone disease. We will assess diet and collect stool and urine from 300 participants ?4 years old without recent antibiotic exposure (150 with calcium kidney stones and 150 matched controls), oversampling younger participants. We will sequence the gut microbiome using shotgun metagenomics and measure downstream metabolites using untargeted metabolomics of stool and urine, targeted short-chain fatty acid metabolomics of stool, and 24-hour urine chemistries. Using novel mediation models, we will define the direct and indirect effect of diet on the gut microbiome and intestinal and urinary metabolites and its contribution to kidney stones. In Aim 2, we will, for the first time, determine the relationship between oral antibiotic exposure and urine chemistries in kidney stone disease. We will link 24-hour urine chemistry results with pharmaceutical claims and clinical data of individuals in the HealthCore database, which includes >48 million individuals. We will conduct a nested case-control study to determine the relationship between the dose and duration of antibiotic exposure and kidney stones and to identify sub-groups at greatest risk. We will then perform a cohort study to identify how oral antibiotics alter urine chemistries. These results will identify metabolites that contribute to kidney stones following perturbation of the gut microbiome and provide key insights for future studies of primary and secondary stone prevention.